Butanols are an important class of chemicals with utility in a wide range of industrial applications, including use as a solvent and reactive intermediate in organic syntheses, as a solvent in the textile industry, as an extractant in the food industry, and as a slow-evaporating solvent for use in enamels and lacquers. Recently butanols (such as isobutanol) are finding increasing use as an alternative to conventional fossil fuels for use in motor vehicles. Unlike ethanol, such butanol fuels are not corrosive, offer an energy density similar to that of gasoline, and can be used as a direct replacement for petroleum-derived fuels in motor vehicles without the need for extensive engine modifications. Branched chain alcohols, such as isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol are particularly attractive in this regard. Currently 10 to 12 billion pounds of butanol are produced annually and needs are projected to increase.
In the United States butanols are generally produced by the hydroformylation of propene to form butyraldehyde, which is subsequently reduced to 1-butanol and 2-butanol. Such process, however, relies on nonrenewable resources and result in the release of greenhouse gases to the environment. As a result methods for the production of butanols have been developed that utilize biological processes and renewable starting materials such as biomass.
Butanol production using biological means, also known as biobutanol, is well known. For example, butanol was noted as a byproduct of the A.B.E. process developed by Chaim Weizmann nearly a century ago, in which acetone is produced by fermentation of starch by Clostridium acetobutylicum. Production of butanol by this process is low, however. More recently a number of investigators have proposed methods for higher yield production of butanols through the use of genetically modified organisms that ferment simple or crude feedstocks. For example, United States Patent Application No. 2009/0081746 (to J. C. Liao et al.) describes use of a variety of genetically modified organisms with mutations in specific metabolic pathways to ferment sugars and produce a variety of higher alcohol biofuels. Similarly, United States Patent Application No. 2009/0288337 (to S. Picatoggio et al.) discloses the use of genetically modified organisms, such as yeasts, to produce methylbutanol biofuel from amino acid precursors. Such approaches, however, necessarily suffer from relatively low yields that limit their use in large scale operations. Such low yields can be due to metabolic bottlenecks inherent in the enzymatic pathways used. For example, in United States Patent Application No. 2008/261230 (to D-I. Liao, M. J. Nelson, and M. G. Bramucci) it was noted that enzymes in these metabolic pathways may be engineered for high expression in a genetically modified organism, but still remain bottlenecks in a fermentative process due to inherent low specific activity for the production of isobutanol. Since isobutanol and similar compounds are relatively toxic it is not clear if enzymes are or will be available with specific activities high enough to support industrial synthesis of butanols at the relatively low concentrations of butanol precursors generated within a living cell. These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
In another approach (as disclosed in U.S. Pat. No. 8,293,509, to S. D. Simpson et al.), a first bioreactor can be used to produce useful alcohols from a crude feedstock and to produce a side product that can be used to enhance the production of butanol from a second, distinct feedstock (such as a carbohydrate) within a second bioreactor. In all of these approaches, however, the inherent toxicity of higher alcohols such as butanol and isobutanol can limit their final concentration in a conventional fermentation process via growth inhibition, metabolic inhibition, and/or death of the productive cells.
Attempts have been made to address the problem of product toxicity in the biological production of butanols. For example, U.S. Pat. No. 8,283,144 (to G. K. Donaldson et al.) describes the use of organisms with resistance (i.e. IC50>0.5% w/v) to isobutanol to produce this product by fermentation of sugars. Similarly, United States Patent Application No. 2007/259411 (to M. G. Bramucci et al) discloses a method for improving the resistance of butanol-producing Enterococcus strains evolutionary means and their subsequent use in the synthesis of butanol and isobutanol. It is not clear, however, if the concentration of these higher alcohols that can be tolerated are economically recoverable at large scale. Another approach is suggested in United States Patent Application No. 2008/274526 (to M. G. Bramucci et al.), in which the temperature of the fermentation process is reduced in order to improve tolerance to the isobutanol product. Such a reduction in temperature, however, both reduces the rate of isobutanol production and can increase energy costs.
Yet another approach to reducing the effect of product toxicity is to provide a means for segregating biologically produced butanols from the sensitive cell population. One method, disclosed in United States Patent Application (No. 2011/097773, to M. C. Grady et al.), is to provide a water-immiscible solvent system that extracts 1-butanol, 2-butanol, and isobutanol from fermentation media. Large scale application of this approach is limited, however, by the need to occupy a relatively large portion of the fermenter volume with the non-productive solvent system. An alternative approach is disclosed in European Patent No. 0,047,641B1 (to H. G. Lawford), in which the fermentation process is split into an initial phase where little alcohol (in this instance ethanol) is produced and cells replicate rapidly and a second phase where growth-inhibiting concentrations of alcohol are produced by the culture amassed during the initial phase by changing culture conditions. Such an approach, however, retains the metabolic limitations of performing a complete fermentative process within a single organism.
Thus there remains a need for efficient and scalable biological synthesis of alcohols, and particularly branched chain butanols, from simple feedstocks.